text:
Verapamil Alleviates Myocardial Ischemia/Reperfusion Injury by Attenuating Oxidative Stress via Activation of SIRT1

Myocardial ischemia/reperfusion (I/R) injury is a potential complication of ischemic heart disease after recanalization. One of the primary reasons for I/R injury is the excessive accumulation of reactive oxygen species (ROS) in cardiomyocytes. Verapamil, a classic calcium channel blocker, has the potential to mitigate I/R-evoked oxidative stress. However, the underlying mechanisms have not been fully elucidated. SIRT1 is an essential regulator of I/R and offers resistance to oxidative stress arising from I/R. It is still inconclusive if verapamil can reduce myocardial I/R-triggered oxidative damage through modulating SIRT1 antioxidant signaling. To verify our hypothesis, the H9c2 cardiomyocytes and the mice were treated with verapamil and then exposed to hypoxia/reoxygenation (H/R) or I/R in the presence or absence of the SIRT1 inhibitor EX527. As expected, verapamil stimulated SIRT1 antioxidant signaling evidenced by upregulation of SIRT1, FoxO1, SOD2 expressions and downregulation of Ac-FoxO1 expression in vitro and in vivo. In addition, verapamil remarkably suppressed H/R and I/R-induced oxidative stress proven by declined ROS level and MDA content. The cardioprotective actions of verapamil via SIRT1 were further confirmed in the experiments with the presence of the specific SIRT1 inhibitor EX527. We demonstrated that verapamil alleviated myocardial I/R-evoked oxidative stress partially via activation of SIRT1 antioxidant signaling. Subsequently, verapamil protected against cardiac dysfunction and myocardial infarction accompanied by oxidative stress.

INTRODUCTION
Myocardial infarction following ischemia is responsible for the high lethality of coronary heart disease (CHD) (Kober, 2017). The current common therapies for patients with severe CHD include coronary artery bypass grafting or percutaneous coronary intervention (Lozano et al., 2020). In fact, restoring blood supply to ischemic myocardium may further aggravate tissue damage, namely myocardial ischemia/reperfusion (I/R) injury (Bi et al., 2018). Myocardial I/R injury is a complicated and multifactorial pathological process (Kawaguchi et al., 2011;Kalogeris et al., 2016;Xie et al., 2021).
Of the many factors regarding the process of myocardial I/R injury, the burst of reactive oxygen species (ROS) during the reperfusion is a pivotal one with extensive well-documentation . The excessive accumulation of ROS induces peroxidation to protein, DNA and lipid and subsequently leads to myocardial apoptosis, as observed in cardiac tissue subjected to I/R operation (Zhai et al., 2017).
While the impairment of oxygen free radicals in the hearts during I/R was recognized, increasing attention was driven in seeking antioxidants as a potential treatment option. Verapamil, a classic calcium channel blocker, plays a critical role in dilating coronary artery and is commonly used to treat angina, arrhythmias and hypertension (Klein and Weiner, 1987;Cummings et al., 1991;Tang et al., 2016). Numerous reports have also revealed that verapamil antagonizes I/R injury in different experimental models such as heart, brain, liver and so on (Korn et al., 1988;Nauta et al., 1991;Jangholi et al., 2020). Further studies have reported that mitigating oxidative stress by verapamil plays a crucial role in ameliorating myocardial I/R injury (Mandal et al., 2007;Mohan et al., 2009). Our prior study has also showed that verapamil exhibits an antioxidant action in both primary cardiomyocytes H/R and rats myocardial I/R models Zhang et al., 2013). However, the underlying mechanisms have not been completely explored.
Silent information regulator of transcription 1 (SIRT1) is a deacetylase that removes the acetyl group from target proteins in the presence of nicotinamide adenine dinucleotide (NAD + ) (Imai et al., 2000;Landry et al., 2000;Yang et al., 2013). SIRT1 is an essential regulator of myocardial I/R injury with multiple actions including resisting oxidative stress (Alcendor et al., 2007), inhibiting apoptosis (Yu et al., 2017), decreasing inflammation (Yu et al., 2016) and protecting mitochondrial function (Yang et al., 2015). Studies have identified that SIRT1 protects against hypoxia/reoxygenation (H/R) or I/R injury by attenuating oxidative stress (Becatti et al., 2012;Zhang et al., 2021). Li et al. suggested that CAPE-oNO 2 suppressed the excessive generation of ROS via modulating the SIRT1/eNOS/NF-κB signal axis in the I/R and H/R models . Han and colleagues also demonstrated that SIRT1 agonism inhibited pyroptosis and further alleviated myocardial I/R injury by modulating pyruvate dehydrogenase-associated glucose oxidative metabolism (Han et al., 2020). Moreover, Hsu et al. identified that in transgenic mice, modest overexpression of SIRT1 resisted myocardial I/R-evoked oxidative stress through deacetylating Ac-FoxO1 to trigger the transcription of SOD (Hsu et al., 2010). However, whether SIRT1/FoxO1 antioxidant signaling participated in verapamil's antioxidant actions against I/R remained unclear.
Therefore, we designed the experiments to explore whether SIRT1 antioxidant signaling was associated with the amelioration of verapamil in myocardial I/R-induced oxidative damage.

Animals
All animal study procedures were conducted in adherence with the National Institutes of Health (NIH publication no. 86-23, revised 1996)

Cell Culture and Establishment of Experimental Models
H9c2 cells obtained from American Type Culture Collection (ATCC, USA) were cultured in DMEM medium with 10% FBS, 100 U/ml of penicillin and 0.1 mg/ml streptomycin in a humidified cell incubator (95% air, 5% CO 2 ) at 37°C. To construct cardiomyocytes H/R model, H9c2 cells were exposed to a hypoxic buffer containing the following reagents (mM): CaCl 2 (0.9), MgCl 2 (0.49), KCl (12), NaCl (137), Na lactate (20) and HEPES (4) at pH 6.2 in a hypoxic incubator (5% CO 2 , 94% N 2 , 1% O 2 ) for 3 h and then reoxygenation was conducted by the replacement with normal air conditions and medium with 0.5% FBS for 1 h (Zhou et al., 2010). Verapamil (0.1, 1, 10 μM) was dissolved in the DMSO and administrated throughout the onset of H/R. To explore whether verapamil alleviated H/R-evoked oxidative stress by agitating SIRT1 antioxidant signaling axis, H9c2 cells were treated with and without 50 μM EX527 for 1 h before hypoxia, respectively.

Preparation of Mouse Myocardial I/R Model and Experimental Design in Vivo
The mouse myocardial I/R model was conducted by ligating the left anterior descending coronary (LAD) following previously detailed description (Gao et al., 2009). In short, the mouse anesthetized with Frontiers in Pharmacology | www.frontiersin.org February 2022 | Volume 13 | Article 822640 sodium pentobarbital (75 mg/kg, intraperitoneally injection) breathed with the aid of the ventilator. Then, we dissected the intercostal muscle of the mouse between the third and the fourth ribs to explore the heart. After accurately locating the LAD, we ligated it with an 8-0 silk suture as well as a small PE-10 polyethylene hose. Following 30 min of coronary occlusion, the heart blood flow was restored for 4 h or 24 h by removing the hose and untying the slipknot. The sham-operated mouse went under the parallel surgery, without the ligation silk tightened. Before the surgery, the mice were given different treatments. The mice were assigned into four groups randomly as following: Sham, I/R, I/R + Ver (1 mg/kg), I/R + Ver + EX527 (5 mg/kg/day, total 3 days before I/R). Verapamil was dissolved in saline and was administrated via intraperitoneal injection 20 min prior to ischemia. EX527 was firstly dissolved in DMSO and then diluted as the final concentration of 1 mg/ml with double distilled water according to the instruction.

Assessment of ROS Generation in H9c2 Cells
The measurement of ROS production in H9c2 cells was carried out by flow cytometry with the DCFH-DA probe. In short, H9c2 cells were incubated in 1 μM DCFH-DA during reperfusion. Afterwards, the cells were rinsed with phosphate buffered saline (PBS) and then digested with trypsin. At last, the cells were rinsed with PBS thrice and examined with the BD Accuri ™ C6 flow cytometry (BD Biosciences, CA, United States) at 525 nm emission wavelength and a 488 nm excitation wavelength.

Determination of ROS Generation in Heart Tissues
The ROS level in myocardial frozen section was detected by DHE. Briefly, the 8 μm-thick tissue sections were incubated with 5 μM DHE at 37°C for 30 min in the dark. Afterwards, the sections were rinsed with PBS thrice. Finally, the ROS level of the tissue sections was observed under Olympus BX53 (Olympus, Tokyo, Japan) microscope. The mean fluorescent intensity (MFI) was quantified by Image-J (NIH, United States).

Oxidative Damage Assessment
MDA, a product of peroxidation, was detected by the detection kit referring to the protocol of manufacture.

Echocardiographic Measurement
Following 4 h of reperfusion, we measured cardiac systolic function of the mice with an ultrasound probe as mentioned previously (Wang et al., 2021). The ultrasound videos and images of mouse hearts were collected by Vevo LAZR photoacoustic imaging system (Fujifilm Visualsonics, Toronto, ON, Canada). Left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were calculated with computerized algorithms.

Myocardial Infarct Size
Myocardial infarction was detected by Evans blue and TTC double staining. After 24 h of reperfusion, the LAD was retied in situ. 0.5 ml of 1% Evans blue dye injected via the right atrium stained the non-ischemic myocardium blue. Subsequently the heart was quickly harvested, washed with saline and frozen at −30°C. As soon as the heart was frozen, it was cut transversely into 1 mm-thick slices and incubated in 1% TTC for 20 min at 37°C. The ischemic but viable myocardium was stained red in TTC dye, while only the infarction area was pale.

Western Blot Analysis
The protein expressions were assessed by western blotting as aforementioned (Wang et al., 2015). In short, the total protein was extracted from H9c2 cells with RIPA lysis buffer and 2% protease inhibitor. The heart tissue was rinsed with PBS for the removal of blood and then homogenized with RIPA lysis buffer and 2% protease inhibitor. Then the heart homogenate was centrifuged at 13,000 rpm for 15 min twice and cell homogenate once at 4°C. Protein supernatant was boiled in SDS-PAGE sample loading buffer for 5 min. Protein concentration was determined with the PierceTM bicinchoninic acid (BCA) protein assay kit. SDS-PAGE was used to separate the proteins, which were then transferred onto nitrocellulose membranes. Following 1 h of blockage with 5% defatted milk, the membranes were incubated with antibodies against SIRT1, Ac-FoxO1, FoxO1, SOD2, β-actin and GAPDH overnight at 4°C. Following washes with TBST thrice, the membranes were probed with secondary antibodies for 90 min. Subsequently, the membranes were rinsed with TBST thrice. At last, the protein bands were visualized with SuperSignal detection kit. The gel-Pro software (Media Cybernetics, United States) was used to analyze the gray value of protein bands for quantification. Densitometry of the bands were normalized to that of housekeeping genes.

Statistical Analysis
Data were expressed as the means ± standard error of mean (SEM). Statistical analyses of differences between groups were carried out using t-test or one-way analysis of variance (ANOVA) followed by least significance difference (LSD). A p value less than 0.05 was considered as statistically significant. The SPSS software package version 19.0 (SPSS, Chicago, IL) was used for data analysis.

Verapamil Agitated SIRT1 Antioxidant Signaling and Suppressed the ROS Overproduction in H9c2 Cells Subjected to H/R
Results for immunoblotting analysis manifested that the protein expressions of SIRT1, the total FoxO1 and SOD2 dropped sharply in the H/R group compared with the control group. On the contrary, verapamil treatment increased SIRT1, the total FoxO1 and SOD2 expressions in a dose-dependent fashion in the H/R conditions. (Figures 1A-D). The efficacy was the most obvious in the group treated with 10 μM verapamil, which was chosen for the following experiments. Studies have revealed that enhanced ROS production and oxidative stress are found in myocardium during I/R (Cuzzocrea et al., 2001;Jin et al., 2017). Therefore, to  Figure 1E). These results suggested that verapamil could stimulate SIRT1, FoxO1 and SOD2 and attenuate oxidative stress in H9c2 cells exposed to H/R.

EX527 Abrogated Verapamil-Induced Stimulation of SIRT1 Antioxidant Signaling and Antioxidant Effects in H9c2 Cells Subjected to H/R
To further clarify the mechanism underlying verapamil's antioxidant effects, a selective SIRT1 inhibitor EX527 was introduced to our experiment. In comparison to verapamiltreated cells, the cells simultaneously treated with EX527 presented in shrinkage or death (Figure 2A). EX527 notably abolished the verapamil-mediated stimulation of SIRT1, the total FoxO1 and decreased the expression of SOD2 ( Figures 2B-E), which validated our hypothesis that verapamil could stimulate SIRT1 antioxidant signaling in the H/R models. We hypothesized that SIRT1 antioxidant signaling was implicated in the antioxidant actions of verapamil in the H/R cardiomyocytes. Flow cytometry detected a remarkable augment in ROS formation in the cells simultaneously treated with EX527, but not in the cells treated with verapamil ( Figure 2F). Taken together, verapamil dramatically reduced the H/R-induced ROS overproduction partly via activation of SIRT1 antioxidant signaling in the H9c2 cells. Subsequently, the action of verapamil on SIRT1 antioxidant signaling was validated in the mice with I/R. The results in the mice were consistent with those obtained in the H9c2 cells. In the mice with I/R, there was observation of a downregulation of SIRT1, the total FoxO1 and SOD2 expressions but an upregulation of Ac-FoxO1 expression. Conversely, verapamil administration apparently promoted SIRT1, the total FoxO1, SOD2 expressions and suppressed Ac-FoxO1 expression ( Figures 3A-E). However, the action of verapamil on SIRT1, the total FoxO1, Ac-FoxO1 and SOD2 expressions was overturned by EX527. In summary, verapamil was capable of promoting the SIRT1 antioxidant signaling in the mice with I/R.
The association between SIRT1 antioxidant signaling and verapamil's antioxidant action in vitro reminded us that SIRT1 may also be correlated with verapamil's antioxidant action in vivo. To prove this speculation, DHE was applied to the measurement of ROS production in cardiac tissue.
Compared to the sham-operated mice, ROS generation was dramatically increased in the mice with I/R. In contrast, verapamil administration obviously reduced the I/R-triggered ROS production ( Figures 4A,B). In addition, the MDA content was measured to estimate the level of lipid peroxidation. I/R induced an apparent upregulation of the MDA content, while verapamil administration downregulated the content of MDA ( Figure 4C). In general, verapamil played an antioxidant role in myocardial I/R. SIRT1 selective inhibitor EX527 repressed the effects of verapamil in the I/R hearts, implying that SIRT1 mediated the antioxidant action of verapamil.

EX527 Blunted the Cardioprotective Effects of Verapamil in Mice With I/R
In order to ascertain that SIRT1 participated in the verapamil-mediated cardioprotective effects in the mice subjected to I/R, we assessed the cardiac function and myocardial infarct size of the mice. After 4 h of reperfusion, the mice with I/R showed a decrease in LVEF and LVFS. On the contrary, echocardiography illustrated an increase of LVEF and LVFS in verapamil treatment group compared with the I/R group (Figures 5A-C). After 24 h of reperfusion, the infarct size in the I/R group increased apparently to 38.5 ± 2.55%. In contrast, verapamil administration decreased the infarct size from 38.5 ± 2.55% to 16.8 ± 3.24% compared with the I/R group (Figures 6A-C).
It was evident that verapamil was able to restore post-I/R cardiac function of the mice. However, EX527 diminished the protective effects. These results suggested that part of verapamil's protective effects was mediated by SIRT1 antioxidant signaling pathway.

DISCUSSION
Our experiments confirmed that verapamil had the potential to reduce oxidative stress and consequently reconstruct cardiac function in the H9c2 cells H/R and the mice I/R models. We revealed a novel molecular mechanism by which verapamil mitigated myocardial I/R-provoked oxidative stress. For the first time, our results demonstrated that verapamil's cardioprotective action against myocardial I/R injury was partly mediated by the stimulation of SIRT1 antioxidant signaling in vitro and in vivo (Figure 7). Oxidative stress, regarded as the primary cause of myocardial I/R injury, initiates a cascade of deleterious cellular processes including endothelial dysfunction, cardiomyocyte injury and apoptosis (Simon et al., 2021). During reperfusion, overproduction of ROS triggers cytosolic Ca 2+ overload, which in turn disrupts the balance of Ca 2+ -dependent redox systems, resulting in a further increase of ROS (Aldakkak et al., 2011;Chang et al., 2019). Excessive ROS forces the mitochondrial permeability transition pore to open, leading to cytochrome c release from the mitochondria and eventually initiating the endogenous apoptosis pathway responsible for cell death and cardiac dysfunction (Murphy and Steenbergen, 2008). In this study, verapamil exhibited a potent antioxidant capability in the H9c2 cells H/R and the mice I/R models that verapamil treatment significantly increased the expression of antioxidant enzyme SOD2 with decreased ROS level and MDA content according to our observation. I/R-evoked massive ROS generation promoted the cardiomyocytes death and cardiac dysfunction with gross increase in the infarct size as well as decrease in LVFS and LVEF. Verapamil treatment significantly reversed this adverse phenomenon.
The sirtuin family are NAD + -dependent deacetylases. SIRT1, the most extensively studied sirtuin, is beneficial in cardiovascular system (Kane and Sinclair, 2018). Currently, it is well accepted that adenosine monophosphate-activated protein kinase (AMPK) is the most common pathway responsible for the activation of the majority in sirtuin family (Eid et al., 2021). AMPK enhances SIRT1 activity by increasing cellular NAD + levels while SIRT1 deacetylates liver kinase B1 (LKB1), leading to phosphorylation and activation  (Lan et al., 2008;Iwabu et al., 2010). In theory, I/R-induced Ca 2+ overload may lead to activation of calcium/calmodulin-dependent protein kinase2 (CaMKK2) (Sung & Choi, 2012) and eventually cause the compensatory elevation of AMPK. Though the results in our study were in contradiction to the theory, presenting the downregulation of SIRT1 during I/R, Park et al. suggested a biphasic control of AMPK activity by Ca 2+ (Park et al., 2011). Moreover, studies showed that the levels of SIRT1 and p-AMPK were significantly decreased in the infarcted myocardium of I/R-induced rats (Eid et al., 2021;Liu et al., 2021), suggesting that Ca 2+ may indirectly influence SIRT1 signaling by regulating AMPK, of which the potential mechanisms awaited to be investigated. It has been reported that mitochondrial Ca 2+ overload rapidly increased protein acetylation via a buildup of NADH and the inhibition of sirtuin enzymatic activity (Marcu et al., 2014). In addition, the increase of Ca 2+ concentration in cytosol activated protein kinase Cδ (PKCδ) which negatively regulated SIRT1 expression . Our previous study revealed that the quaternary ammonium salt derivative of haloperidol, N-n-Butyl haloperidol iodide (F 2 ), a new calcium antagonist that reduced cytosolic calcium level, inhibited the Ca 2+ -dependent translation of PKCα . In the present study, verapamil, a calcium channel blocker, induced the activation of SIRT1 likely via the elevation of [NAD + /NADH] cyt ratio or the inhibition of PKC. However, the exact mechanism of SIRT1 upregulation in verapamil treatment group would be further studied in the future.
SIRT1 eliminates ROS and maintains the balance of redox system in mitochondria mainly by deacetylating Ac-FoxO1 and promoting the transcription of antioxidant genes including SOD, catalase and GSH-Px. Previously, it has been established that the deacetylation of Ac-FoxO1 via SIRT1 enabled FoxO1 to initiate the transcription of SOD, thereby counteracting ROS evoked by I/R (Hsu et al., 2010). Besides, curcumin-induced activation of SIRT1/FoxO1 signaling and diminution of mitochondrial oxidative damage showed protective effects in rat I/R hearts and H/R cardiomyocytes (Yang et al., 2013). In the current study, we discovered that verapamil potentiated SIRT1 expression, accompanied by the low expression of Ac-FoxO1 in I/R. Importantly, upregulation of SIRT1 induced by verapamil increased the expression of SOD2 which contributed to the reduction of I/R-evoked oxidative stress. Verapamil reduced oxidative stress and thereby protected the mice against myocardial I/R injury reflected by restored cardiac function and decreased infarct size. Notably, inhibiting SIRT1 by EX527 remarkably blunted the cardioprotective actions of verapamil in I/R. These results suggested that verapamil-induced stimulation of SIRT1 downregulated the acetylation of FoxO1 and exerted resistance to oxidative damage in I/R hearts.
Beyond that, we were surprised to observe that H/R and I/R could induce the downregulation of the total FoxO1 expression. Conversely, verapamil apparently enhanced the total FoxO1 expression in H/R and I/R. The results from EX527 and verapamil treated groups again supported our findings. Interestingly, verapamil downregulated Ac-FoxO1 expression but enhanced SIRT1-dependent expression of the total FoxO1 in I/R. Hsu et al. identified that the total FoxO1 was positively regulated by SIRT1 not only at the protein level but also at the mRNA level in cultured cardiomyocytes. Verapamil may augment SIRT1-dependent expression of the total FoxO1 largely by strengthening the transcription of FoxO1. Subsequently, FoxO1 could induce the expression of SOD and thereby mediate the antioxidant actions of verapamil in H/R and I/R. We verified that the stimulation of SIRT1/FoxO1 signaling pathway contributed to the beneficial effects of verapamil in myocardial I/R injury. However, the exact performance of different postmodification of FoxO1 in I/R was not elucidated. Further work was demanding to clarify the precise relationship among the total FoxO1, SIRT1 and I/R. There are still some limitations in our study. Firstly, our in vitro experiments were performed in H9c2 cells which may not be able to fully mimic the function of neonatal cardiomyocytes. Secondly, the conclusion that the cardioprotective effects of verapamil in I/R were partly mediated by SIRT1 was verified using EX527. EX527 may have some inhibitory effects on SIRT2, but its selectivity is much lower than that on SIRT1. Still, the specificity of EX527 and the inhibition mechanisms of EX527 are under investigation. Despite these limitations, we believe that this study has provided important new information for the understanding of antioxidant effects of verapamil in H/R and I/R.

CONCLUSION
In conclusion, our results confirm that verapamil alleviates myocardial I/R injury by inhibiting the oxidative stress in both in vivo and in vitro models. In addition, we further confirm that the cardioprotective effects of verapamil are antagonized in the presence of EX527. All of the results suggest that the effects of verapamil against I/R injury are closely related to the activation of SIRT1 antioxidant signaling. Thus, our study provides a new mechanism for verapamil in the treatment of ischemic heart disease.


A Transfectable Fusagravirus from a Japanese Strain of Cryphonectria carpinicola with Spherical Particles

A novel dsRNA virus (Cryphonectria carpinicola fusagravirus 1, CcFGV1), isolated from a Japanese strain (JS13) of Cryphonectria carpinicola, was thoroughly characterized. The biological comparison of a set of isogenic CcFGV1-infected and -free (JS13VF) strains indicated asymptomatic infection by CcFGV1. The sequence analysis showed that the virus has a two open reading frame (ORF) genome of 9.6 kbp with the RNA-directed RNA polymerase domain encoded by ORF2. The N-terminal sequencing and peptide mass fingerprinting showed an N-terminally processed or degraded product (150 kDa) of the 5′-proximal ORF1-encoded protein (1462 amino acids) to make up the CcFGV1 spherical particles of ~40 nm in diameter. Interestingly, a portion of CcFGV1 dsRNA co-fractionated with a host protein of 70 kDa. The purified CcFGV1 particles were used to transfect protoplasts of JS13VF as well as the standard strain of an experimental model filamentous fungal host Cryphonectria parasitica. CcFGV1 was confirmed to be associated with asymptomatic infection of both fungi. RNA silencing was shown to target the virus in C. parasitica, resulting in reduced CcFGV1 accumulation by comparing the CcFGV1 content between RNA silencing-competent and -deficient strains. These results indicate the transfectability of spherical particles of a fusagravirus associated with asymptomatic infection.

There are few reports on the biological characterization of fungal fusagraviruses, largely because of the lack of methods for their experimental introduction. Therefore, no viral etiology is established for many fusagravirus infections. This contrasts with many other fungal dsRNA viruses such as the members of orders Durnavirales (partitiviruses), Ghabrivirales (victoriviruses, megabirnaviruses, chrysoviruses, and megatotiviruses), and Reovirales (mycoreoviruses), respectively, for which virion transfection has been developed [15][16][17][18][19][20]. Related to this drawback is the ambiguity as to whether fusagraviruses are capsidless or encapsidated dsRNA viruses. Spear et al. suggested the capsidless nature of insect fusagra-like viruses [12], whereas plant fusagraviruses have been shown to encode capsid protein on the 5 -proximal ORF [11]. In contrast to many other fungal dsRNA viruses, a capsidless nature was also suggested for a few fungal fusagraviruses [2,5]. In this sense, we have previously developed a method to examine whether a virus of interest is capsidless, associated with any host proteins, protected by host-derived membrane vesicles, or encapsidated [21,22].
The genus Cryphonectria contains an important plant pathogen, Cryphonectria parasitica, causing one of the three most destructive tree diseases in addition to other plant pathogenic and non-pathogenic fungi [23][24][25]. It is noteworthy that C. parasitica serves as a model filamentous fungal host suitable for studying host antiviral RNA silencing/viral counterdefense responses [26,27]. These Cryphonectria spp. are often sympatric to each other. C. parasitica, in particular, has been studied extensively from perspectives of biocontrol research, phytopathology, and virology [23,26]. Several research groups have searched large collections of C. parasitica isolates from different localities and discovered a variety of viruses [28][29][30][31]. Cryphonectria nitschkei and Cryphonectria naterciae, much less studied than C. parasitica, were also found to naturally host several viruses [9,16,32]. Milgroom and colleagues screened a collection of C. parasitica and C. nitschkei collected from Japan and China and discovered unidentified RNA viruses [28]. Among them, a Japanese strain, JS13, was originally identified as C. nitschkei, and the authors claimed it to be the strain infected with an RNA virus unrelated to hypoviruses most prevailing in C. parasitica (family Hypoviridae, well-known Cryphonectria viruses with a positive-sense, single-stranded (+)ssRNA genome) [33]. This fungal strain was re-classified as Cryphonectria carpinicola (see Figure 1), which is a recently established species within the genus Cryphonectria [25].
In the current study, we report on the thorough characterization of a novel fusagravirus, termed Cryhonectria carpinicola fusgravirus 1 (CcFGV1), from strain JS13 of C. carpinicola. Furthermore, we developed a transfection method with spherical particles of CcFGV1, which allowed us to conclude its asymptomatic nature in C. parasitica as well as in the original host.

Sequence Determination of CcFGV1 and Northern Blotting
CcFGV1 strain isolated from C. carpinicola JS13 was partially characterized by Liu et al. [28]. The virus was described a non-hypo virus based on the observation that the CcFGV1 genomic dsRNA could not hybridize with any of CHV1-, CHV2-, CHV3-, or CHV4-specific probes in northern blotting. In this study, dsRNA was purified from strain C. carpinicola JS13 and used as a template for cDNA library construction using a non-PCR-based conventional method, as described by Lin et al. [39]. After determining the sequences of cDNA clones (at least three clones with the same region were sequenced), we designed primer sets for 3 -RNA ligase-mediated rapid amplification of cDNA ends (3 -RLM-RACE). The complete nucleotide sequence of the CcFGV1 genome was deposited in the GenBank/DDBJ/ENA database under accession number LC651180. The sequences of oligonucleotides used in RACE are listed in Supplementary Table S1.
Total RNA was purified from PDA-cellophane cultures as described by Eusebio-Cope and Suzuki [40], electrophoresed in agarose gel, and denatured in alkaline conditions. Specific detection of CcFGV1 RNA was carried out according to the method of Sato et al. [22] using a digoxigenin (DIG)-labeled PCR-amplified DNA probe (Table S1).

Preparation of Polyclonal Antibodies to CcFGV1 ORF1-Encoded Protein and Western Blotting
To prepare antibodies against the CcFGV1 ORF1-encoded protein (150 kDa protein), the N-and C-terminal, 300 amino acids were over-expressed in Escherichia coli as glutathione S-transferase (GST) fusion products using an expression vector, pCold (Takara Bio Inc., Otsu, Japan). The native form of both the fusion proteins was purified by GST chromatography according to the manufacturer's instructions after being highly expressed at 15 • C. A denatured form of both the fusion proteins was purified from preparative SDSpolyacrylamide gel electrophoresis (SDS-PAGE) gel as described earlier. Equal amounts of the N-terminal (denatured) and C-terminal (native and denatured) polypeptides were mixed, and the final concentration was adjusted to 1 mg/mL. Similarly, denatured recombinant protein mixtures were prepared. These preparations were injected into one New Zealand white rabbit (Oryctolagus cuniculus) six times at a two-week interval, after being mixed with complete (first injection) and incomplete adjuvants (subsequent five injections).
Western blotting was carried out as described earlier [21]. Proteins were fractionated in 10% SDS-PAGE gel and transferred to a polyvinylidene difluoride (PVDF) membrane (Immobilon-P, Merck Millipore, Billerica, MA, USA). The blot was treated with anti-150 kDa protein antiserum and 1000-fold diluted, mouse anti-rabbit IgG conjugated with horseradish peroxidase, HRP (formerly Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD. USA). Specific interactions were visualized using ECL Prime Western Blotting Detection Reagent (Cytiva Amersham, Amersham, UK) and a LAS4000 chemical luminescence detection system.

Virion Purification, Protein Analyses, and Transfection
CcFGV1-infected mycelia (approximately 10 g, fresh weight) from JS13 was harvested (from 10-day-old cellophane-PDA culture), ground to a fine powder in the presence of liquid nitrogen, and homogenized in 40 mL of 0.1 M sodium phosphate buffer (PB), pH 7.0, 40 µL of 2-mercapto ethanol, and 1/4 the volume of CCl 4 . This homogenate was centrifuged at 3.5 kilo-rotations per minute (krpm) in a KUBOTA RS-240 rotor (Kubota Co., Tokyo, Japan) for 10 min. The supernatant was centrifuged at 36 krpm in a Beckman 70Ti rotor for 1.5 h. The pellet was resuspended in up to 1 mL of 0.05M PB and used as the crude particle fraction (CPF) in transfection (see below). The CPF was subjected to sucrose density gradient (SGD, 10-50%) centrifugation at 36 krpm for 1.5 h for further particle purification in a Beckman SW41Ti swing rotor. The gradient was fractionated (12 per tube), diluted with about 3 volumes of 0.05 M PB, and ultracentrifuged at 36 krpm for 1.5 h. After being resuspended in 100 µL of 0.05 M PB, each fraction was analyzed for RNA and protein components or subjected to electron microscopy and transfection. Purified particles were negatively stained with an EM stain (Nissin EM Co., Tokyo, Japan) [47] and observed using a model H-7650 transmission electron microscope (Hitachi, Tokyo, Japan).
Peptide mass fingerprinting (MS/MS analysis) was carried out at the Department of Instrumental Analysis and Cryogenics, Advanced Science Research Center, Okayama University (Okayama, Japan). Proteins in CcFGV1 fractions were loaded for 10% SDS-PAGE and the gel was stained with the Rapid Stain CBB Kit (Nacalai Tesque Inc., Kyoto, Japan) or SilverQuest TM Silver Staining Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA), while dsRNA was analyzed by agarose gel electrophoresis in the 0.5× TAE buffer system. Each protein band of interest was subjected to in-gel tryptic digestion according to the manufacturer's instructions (Bruker Daltonics Inc., Billerica, MA, USA). The digested peptides were subjected to LC-MS/MS analysis with the HPLC-Chip/QTOF system (Agilent Technologies Inc., Santa Clara, CA, USA) at the Department of Instrumental Analysis and Cryogenics, Okayama University, and identified using Mascot software (Matrix Science Inc., Boston, MA, USA).
For N-terminal amino acid sequencing, proteins separated on an SDS-PAGE gel were transferred onto a PVDF membrane, followed by amido black staining. The CcFGV1 150 kDa band (see Section 3) was then excised from the membrane and processed for amino acid sequences on a gas-phase protein sequencer Shimadzu Model PPSQ-31A (Kyoto, Japan) at the Department of Instrumental Analysis and Cryogenics, Okayama University.

RNase A Assay
The RNase assay was performed according to the method of Sato et al. [22] with a slight modification. Fungal mycelia were homogenized in four volumes (v/w) of 0.05 M PB, after grinding in the presence of liquid N 2 , followed by the removal of mycelial debris using CCl 4 . The homogenate was treated with or without RNase A (10 µg/mL) (Sigma-Aldrich Co. LLC, Milwaukee, WI, USA) for 30 min at 37 • C. RNase A was inactivated with phenol, followed by phenol/chloroform/isoamyl alcohol (25:24:1) and chloroform/isoamyl alcohol (24:1) treatment. DsRNA in the aqueous phase was isolated by cellulose column chromatography, precipitated with iso-propanol, and then subjected to agarose gel electrophoresis.

Asymptomatic Infection of C. carpinicola by CcFGV1
An isolate (JS13VF) judged as a CcFGV1-free strain by dsRNA gel electrophoresis, and RT-PCR was obtained from the originally CcFGV1-infected JS13 strain of C. carpinicola via single spore isolation ( Figure 1A). Of 43 single conidial isolates tested for the presence of CcFGV1 dsRNA, only one tested negative for CcFGV1 when examined by dsRNA gel electrophoretic analysis ( Figure 1B) and colony-direct one-step RT-PCR (data not shown). As reported by Liu et al. [28], strain JS13 was found to carry dsRNA of approximately 10 kbp. A comparison of the isogenic JS13 and JS13VF suggested that CcFGV1 causes asymptomatic infection in C. carpinicola. The two strains showed indistinguishable phenotypes three and seven days post-inoculation onto PDA. The older cultures produced orange pigments ( Figure 1A) much more than the younger ones or the standard C. parasitica EP155 strain (data not shown).

Molecular Properties of the CcFGV1 Genome
Thirty-six independent cDNA clones, which had over 1.5 kbp inserts, were selected from the library, and subjected to Sanger sequencing. Consequently, a long contig of approximately 9 kbp was obtained after assembly. From the size of the CcFGV1 genomic dsRNA expected from agarose gel electrophoresis ( Figure 1B), the contig appeared to span near the full-length genomic sequence. Thus, the remaining terminal sequences were determined by the RACE method, in which at least five independent RACE clones were sequenced. The dsRNA genome of CcFGV1 strain JS13 is 9651 bp in length, with two large ORFs encoding 1462 and 1319 amino acids, respectively. The protein encoded by ORF2 possesses the RdRP domains ( Figure 1C). The CcFGV1 genome has a 5 -AAAAAAC-3 heptamer immediately upstream of the termination codon of ORF1 at map positions 5551-5557. This heptamer is expected to mediate −1 ribosomal frameshifting as predicted for other members of some groups within the order Ghabrivirales [49]. Another non-canonical translation is predicted to occur for ORF1, based on the long 5 -untranslated region of 1171 nucleotides with over 10 small ORFs (6~204 nt in size). Internal Ribosomal Entry Site (IRES)-mediated translation is most likely involved in this virus as for other fungal dsRNA and (+)ssRNA viruses [50].
The BLASTp search showed that the ORF1-and ORF2-encoded proteins share the highest amino acid sequence identity (approximately 50%, e-value 0) to those of Cryphonectria naterciae fusagravirus 1 (CnFGV1) (Tables S2 and S3). There were lower levels of amino acid sequences similarity detected between the two CcFGV1 proteins and the counterparts of other previously reported fusagraviruses such as Fusarium virguliforme dsRNA mycovirus 1 (FvMV1) and Trichoderma atroviride mycovirus 1 (TaMV1) (Tables S2 and S3).

Phylogenetic Analysis of Fusagraviruses
To understand the phylogenetic relationships between CcFGV1 and other fusagraviruses, an ML tree based on RdRP (ORF2 protein) sequences was constructed (Figure 2). The analysis confirmed that CcFGV1 and other fusagraviruses formed a clade with a strong statistical support value (100%) and were distantly related to a group of insect-associated viruses and a plant virus infecting persimmon. CcFGV1 sub-clustered with fusagraviruses from C. naterciae (CnFGV1), F. virguliforme (FvMV1 and FvMV2), and T. atroviride (TaMV1). This relationship was congruent with the above-mentioned BLASTp result using CcFGV1 ORF2 protein as the query. A similar topology was also observed in the ML tree based on the ORF1-encoded proteins (data not shown).

CcFGV1 dsRNA Is Inaccessible by RNase in Mycelial Homogenate
Whether CcFGV1 formed rigid particles was an interesting question, given that several previously isolated fusagraviruses were reported to be difficult to purify as particles. In order to examine the possibility of the capsidless nature of CcFGV1, we performed an RNase assay developed by Sato et al. [22] for predicting the virus form in mycelial homogenates. We included two fungal RNA viruses: an encapsidated dsRNA virus (a betapartitivirus, RnPV11) and a capsidless (+) ssRNA virus (a hadakavirus, HadV1). The

CcFGV1 dsRNA Is Inaccessible by RNase in Mycelial Homogenate
Whether CcFGV1 formed rigid particles was an interesting question, given that several previously isolated fusagraviruses were reported to be difficult to purify as particles. In order to examine the possibility of the capsidless nature of CcFGV1, we performed an RNase assay developed by Sato et al. [22] for predicting the virus form in mycelial homogenates. We included two fungal RNA viruses: an encapsidated dsRNA virus (a betapartitivirus, RnPV11) and a capsidless (+) ssRNA virus (a hadakavirus, HadV1). The HadV1 dsRNA replicative form was fully digested at 37 • C within 30 min regardless of the addition of RNase A, suggesting that the HadV1 dsRNA replicative form is susceptible to RNase intrinsically present in the host fungus ( Figure 3A). RnPV11 genomic dsRNA was encapsidated and was resistant to RNase in mycelial homogenates. CcFGV1 dsRNA behaved in a way similar to RnPV11 genomic dsRNA ( Figure 3A), and was found to be present in a form resistant to RNase A. HadV1 dsRNA replicative form was fully digested at 37°C within 30 min regardless of the addition of RNase A, suggesting that the HadV1 dsRNA replicative form is susceptible to RNase intrinsically present in the host fungus ( Figure 3A). RnPV11 genomic dsRNA was encapsidated and was resistant to RNase in mycelial homogenates. CcFGV1 dsRNA behaved in a way similar to RnPV11 genomic dsRNA ( Figure 3A), and was found to be present in a form resistant to RNase A. To examine whether CcFGV1 was enclosed in cytoplasmic lipid vesicles like hypoviruses [51,52], we treated the homogenates with the non-ionic detergent Triton X-100. Consequently, CHV1 dsRNA replicative form became susceptible to RNase A only after treatment with Triton X-100, whereas CcFGV1 genomic dsRNA remained resistant even after being treated with Triton X-100, as in the case for the genomic dsRNA of RnPV11 ( Figure 3B and data not shown).
These results suggest that CcFGV1 is encapsidated like other classical dsRNA viruses or tightly associated with proteins like polymycoviruses (multi-segmented dsRNA viruses distantly related to some animal (+)ssRNA viruses such as caliciviruses) [21,53]. This possibility was tested in the following subdivision. This is worthy of note that both encapsidated dsRNA viruses and polymycoviruses can be pelleted by ultracentrifugation, and these pelleted fractions are infectious without any further purification [53].

Particle Fractionation and Identification of Capsid Protein-Coding Region
We attempted to purify CcFGV1 particles from the virus-infected strain JS13 of C. carpinicola by conventional differential centrifugation and SDG centrifugation. Fractions were monitored for the CcFGV1 genomic dsRNA and protein components after ultracentrifugation. These fractions were subjected to dsRNA and protein analyses, transmission electron microscopy, and transfection of C. parasitica. Interestingly, agarose gel electrophoresis showed that dsRNA-rich fractions (fractions 2-4) were observed in a relatively upper zone of the SDG, while the presence of dsRNA was also detected in the fractions To examine whether CcFGV1 was enclosed in cytoplasmic lipid vesicles like hypoviruses [51,52], we treated the homogenates with the non-ionic detergent Triton X-100. Consequently, CHV1 dsRNA replicative form became susceptible to RNase A only after treatment with Triton X-100, whereas CcFGV1 genomic dsRNA remained resistant even after being treated with Triton X-100, as in the case for the genomic dsRNA of RnPV11 ( Figure 3B and data not shown).
These results suggest that CcFGV1 is encapsidated like other classical dsRNA viruses or tightly associated with proteins like polymycoviruses (multi-segmented dsRNA viruses distantly related to some animal (+)ssRNA viruses such as caliciviruses) [21,53]. This possibility was tested in the following subdivision. This is worthy of note that both encapsidated dsRNA viruses and polymycoviruses can be pelleted by ultracentrifugation, and these pelleted fractions are infectious without any further purification [53].

Particle Fractionation and Identification of Capsid Protein-Coding Region
We attempted to purify CcFGV1 particles from the virus-infected strain JS13 of C. carpinicola by conventional differential centrifugation and SDG centrifugation. Fractions were monitored for the CcFGV1 genomic dsRNA and protein components after ultracentrifugation. These fractions were subjected to dsRNA and protein analyses, transmission electron microscopy, and transfection of C. parasitica. Interestingly, agarose gel electrophore-sis showed that dsRNA-rich fractions (fractions 2-4) were observed in a relatively upper zone of the SDG, while the presence of dsRNA was also detected in the fractions from the middle to the bottom portion (fractions 9-12) ( Figure 4A,B). Note that a major peak was observed only in the middle portion of the fraction for the encapsidated partitivirus, RnPV11 (data not shown). SDS-PAGE analysis showed that the upper dsRNA-rich zones (fractions 2-4) also contained a major protein band of 70 kDa, while proteins of 100 kDa and 150 kDa were observed in the middle to the bottom zones, including minor dsRNA-containing fractions (fractions 8-11) ( Figure 4C). In order to investigate whether the detected proteins were virally encoded, we performed western blotting with an antiserum against two recombinant fragments of ORF1-encoded protein ( Figure 1C). As a result, only the 100 kDa and 150 kDa proteins were detected in fractions from the middle to the bottom portion, but not the 70 kDa protein in the upper top fractions ( Figure 4C,D).
Electron microscopy revealed spherical particles of approximately 40 nm in diameter in fraction 9 ( Figure 4E). However, no such particles were observed in fraction no. 3, which corresponded to the upper dsRNA peak (data not shown). Interestingly, two CcFGV1 dsRNA-containing zones had different SDS-PAGE profiles, but virus-like particles were only detected in the lower dsRNA zone ( Figure 4B). We used the same buffer in RNase A treatment as that for virus particle purification. Therefore, the RNase-resistant nature of CcFGV1 genomic dsRNA (Figure 3) can be partially explained by the result of virion purification. Some portion of CcFGV1 dsRNA in the partially purified preparations was a non-virion form, suggesting that the virus may exist in an RNase A-resistant form possibly associated with the 70 kDa protein, together with a classical virion.
Western blotting revealed that the 100 kDa and 150 kDa proteins were CcFGV1 ORF1encoded proteins. We carried out peptide fingerprinting and a chemical determination of the terminal amino acid sequences. MS/MS analysis of the 100 and 150 kDa proteins showed that peptide fragments derived from these proteins were mapped to the C-terminal at two-thirds of the ORF1-encoded protein ( Figure 1C). Peptide mapping patterns for the 100 and 150 kDa proteins ( Figure S1) suggested that the 100 kDa protein was a C-terminally truncated version of the 150 kDa protein. We could determine the N terminal amino acid sequence of the 150 kDa protein to be QFSPSPE-, which was mapped to map positions 264 to 270 of the ORF1-encoded protein.
Taken together, these results suggest that CcFGV1 form spherical particles, the capsid protein of which is encoded by ORF1. Whether the 100 kDa protein is derived by programmed processing or degradation at their C terminus is unclear. This capsid proteincoding profile of CcFGV1 is similar to yado-nushi virus 1 (a toti-like dsRNA virus, the proposed family "Yadonushiviridae"), the capsid protein N-terminus of which starts at around one-third of the ORF1-encoded protein [54].

Purified CcFGV1 Particles Are Trasnfectable and Induce Asymptomatic Infection in Other Cryphonectria Species
Virus transfection was performed on a dsRNA-cured C. carpinicola strain JS13VF, and a strain of the different Cryphonectria species, C. parasitica EP155, and its RNA silencingdeficient mutant ∆dcl2. For virus sources, crude particle preparations, deproteinized purified CcFGV1 dsRNA, and SDG fractions ( Figure 4) were used. The results are summarized in Table 1. Crude extracts inefficiently provided transfectants, while purified CcFGV1 dsRNA failed to show transfectability. Comparison of the fractions 3 and 4 (dsRNA-rich zone, but no virions) as well as 9 (minor dsRNA peak with virions) obtained by SDG centrifugation ( Figure 4) showed that, whereas no transfectants were obtained with fractions 3 and 4, fraction 9 provided a high frequency of transfection of C. carpinicola. Particle fractions prepared by cesium chloride equilibrium centrifugation also gave transfectants, albeit inefficiently, compared to SDG fraction 9 (data not shown).
Transfection allowed for phenotypic comparison between isogenic CcFGV1-free and CcFGV1-infected fungal strains. Figure 5A shows two sets of such fungal strains. As in the case for C. carpinicola, no phenotypic alterations by CcFGV1 were observed in C. parasitica standard strain EP155 and the RNA silencing-deficient mutant ∆dcl2.
We also compared the virus content in three different fungal strains. In C. parasitica EP155, a much lower level of CcFGV1 dsRNA (almost invisible in the total RNA on the agarose gel) was compared to that in C. parasitica ∆dcl2 or C. carpinicola JS13 ( Figure 5B and data not shown). If the latter two were compared, JS13 provided a slightly thicker dsRNA band. Northern blotting with total RNA fractions confirmed a CcFGV1 RNA accumulation pattern similar to the CcFGV1 dsRNA accumulation pattern shown by the agarose gel electrophoresis ( Figure 5B). *, ** Crude virus particle fractions were obtained from mycelial homogenates by centrifugation at 11 krpm for 20 min after stirring at 4 • C in 8% polyethylene glycol 6000 and 1% NaCl for 2 (*) or by one round of differential centrifugation (**) (see Section 2). *** See Virus-free CcFGV1-infected CcFGV1-infected Virus-free Figure 5. Transfection of C. parasitica and CcFGV1 accumulation in the new fungal host strains. C. parasitica wild-type strain (EP155) and an RNA silencing-deficient strain ∆dcl2 were transfected by CcFGV1 crude or purified preparations, or purified dsRNA fractions (Table 1). Two sets of isogenic CcFGV1-free and CcFGV1-infected strains were grown on PDA on the benchtop for five days and photographed (A). (B) Viral accumulation was compared between three fungal strains (C. parasitica EP155 and ∆dcl2, and C. carpinicola JS13) by agarose gel electrophoresis (top panel) and northern blotting (bottom panel).

Discussion
The first near-complete or complete fusagravirus genome sequences were reported for PgLV2 and FgV3 from filamentous fungi, Phlebiopsis gigantea and Fusarium graminearum, in 2009, respectively [2,4]. The number of fusagraviruses genome sequences has grown since then, and this led Wang et al. [1] to propose a new family "Fusagraviridae," which includes not only fungal viruses, but also other related viruses from plants and oomycetes ( Figure 2). However, several virological key questions have been unanswered, such as whether they are encapsidated or what their impact on the host organisms? This study reveals tentative taxonomical placement of, and provides a thorough characterization of a fusagravirus, CcFGV1, and important insights into its virological properties. The closest relative of CcFGV1 is a fusagravirus from C. naterciae, associated with a canker disease of cork oak and chestnut in Europe [24]. The family "Fusagraviridae" has not yet officially been approved, and neither family nor species demarcation criteria have been established by the International Committee on Taxonomy of Viruses (ICTV). However, we propose that these viruses belong to two different species based on the RdRP amino acid sequence identity (45.8%, BLASTp) (Table S3) and phylogenetic relationship (Figure 2). Spherical particles have been purified only for a plant fusagravirus, papaya meleira virus, whose coat protein is encoded by ORF1 [11]. A fungal fusagravirus (TaMV1) was also shown to form isometrical particles of 40 nm in diameter [55]. However, difficulties with particle isolation were noted earlier for fungal fusagraviruses, as well as oomycetous and insect fusagra-like viruses such as PgV2, PiRV3, and SpFV1 [2,10,12]. A capsidless nature is not so unusual for fungal viruses, particularly for (+)ssRNA fungal viruses exemplified by hypoviruses, endornaviruses, narnaviruses, hadakaviruses, and some flexiviruses, although a capsidless nature has not been well-established for dsRNA viruses except for polymycoviruses. It should be emphasized that polymycoviruses form either non-conventional virions (dsRNA-protein complex) or filamentous particles [56,57]. The polymycovirus genomic dsRNA is tightly associated with a virally encoded proline, alanine, and serine-rich protein (PASrp). This form of dsRNA/PASrp is infectious when introduced into host protoplasts [53]. A high content of these three amino acid residues (proline, alanine, and serine) was also noted for the ORF1-encoded protein of PgV2, as observed for capsidless polymycoviruses [53]. These considerations prompted us to suspect a capsidless nature of CcFGV1 at the beginning of this study.
Our RNase assay showed that CcFGV1 dsRNA is in a nuclease inaccessible form in the mycelial homogenates even after treatment with Triton X-100 ( Figure 3B). This suggests two possibilities that (1) CcFGV1 dsRNA is encapsidated; or (2) CcFGV1 dsRNA is tightly associated with protein, as in the case for polymycoviruses. Interestingly, there were two peaks for CcFGV1 dsRNA after SDG centrifugation: one near the top of the gradient and the other peak the two-thirds down. The fraction corresponding to the minor dsRNA peak contained infectious spherical particles of~40 nm in diameter ( Figure 4E) made up of mature ORF1-encoded protein ( Figure 4C,D). A portion of CcFGV1 dsRNA appeared not to be packaged into spherical particles, and was likely associated with the 70 kDa protein ( Figure 4C,D). It is noteworthy that the 70 kDa protein shares sequence identities with cellular alcohol oxidase (S. Hisano and N. Suzuki, unpublished data). Its Helminthosporium victoriae ortholog (Hv-p68) was shown earlier to copurify with the genomic dsRNA of a chrysovirus, Helminthosporium victoriae 145S virus, and possesses RNA-binding activities [58]. If the major fraction of dsRNA was derived from the disruption of particles during purification, the dsRNA would be RNase A susceptible. This unusual fractionation pattern of CcFGV1 may explain the difficulty in purifying fusagravirus particles. Generally, phosphate buffer of an approximately neutral pH has frequently been used for the particle extraction of fungal viruses from filamentous fungi [39,59,60]. However, we found that borate buffer at pH 8.0 provided a much better yield of CcFGV1 (S. Hisano and N. Suzuki, unpublished data). Although we cannot rule out the possibility of the capsidless nature of other fusagraviruses reported to be difficult to purify, it is worth trying different purification methods.
The experimental introduction of fungal viruses is necessary for establishing a viral etiology or determining virus effects on host fungal strains. However, this had never been possible for fusagra-or fusagura-like viruses, and no biological outcome of these dsRNA virus infections has been reported. The only exception was a fusagravirus (CnFGV1) from C. naterciae, which was shown to cause growth defects in strains of a different Cryphonectria species (C. carpinicola) through cross-species horizontal transfer via co-culturing [9]. In this study, CcFGV1 from C. carpinicola was introduced by virion transfection into the strains of two different Cryphonectria species, C. carpinicola and C. parasitica. Virion transfection has become applicable for diverse dsRNA viruses, including chrysoviruses [16], victoriviruses [18,19], yadonushiviruses [54], botybirnaviruses [61] and megabirnaviruses [14] (Ghabrivirales), and partitiviruses (Durnavirales) [15,62], since the first successful attempt with a mycoreovirus (Reovirales) [63]. This study added another family member that can be used in transfection for horizontal transfer. This transfection method might allow for introduction into any fungal host as long as their protoplasts are available. Unlike CnFGV1, CcFGV1 showed asymptomatic infection in the wild-type strains of the two Cryphonectria species (Figures 1 and 5), despite the fact that ∆dcl2 of C. parasitica is deficient in antiviral RNA silencing and is often vulnerable to virus infections [35,64].
Another interesting finding was that even the antiviral RNA silencing-defective C. parasitica strain with the EP155 background (∆dcl2) showed no symptoms upon infection by CcFGV1. Generally, even when RNA silencing-competent C. parasitica EP155 shows little or mild symptoms upon infection of a virus, pronounced symptoms are induced in ∆dcl2 by the same virus, as exemplified by (+)ssRNA and dsRNA viruses (e.g., [14,18,36,65]). Thus, the observation shown in Figure 5 is unusual. Thus far, only a few examples have been reported in which symptomless infection was observed in C. parasitica ∆dcl2 despite the fact that a virus of interest is targeted by RNA silencing and its replication is enhanced in ∆dcl2 as for CcFGV1. Examples include a hypovirus (CHV4) [20] and two Rosellinia necatrix partitiviruses (RnPV3 and RnPV18) [36]. Moreover, there is no phenotypic difference between EP155 and ∆dcl2 infected by Cryphonectria parasitica mitovirus 1 (CpMV1, a mitochondrially replicating virus) [66]. Although CpMV1-derived small RNAs are detected in CpMV1-infected EP155, there is no significant difference in CpMV1 content between EP155 and ∆dcl2 [66]. Thus, it is unclear whether RNA silencing works to inhibit CpMV1 replication.
Fusagraviruses are often co-detected with other RNA viruses in single host fungi [5,7,32,67,68] in which the possible impacts of fusagraviruses on host fungi and coinfecting viruses are unexplored. This study provides a platform for studying virus-host and virus-virus interactions involving fusagraviruses.


High-resolution human KIR genotyping

Killer immunoglobulin-like receptors (KIR) regulate the function of natural killer cells through interactions with various ligands on the surface of cells, thereby determining whether natural killer (NK) cells are to be activated or inhibited from killing the cell being interrogated. The genes encoding these proteins display extensive variation through variable gene content, copy number and allele polymorphism. The combination of KIR genes and their ligands is implicated in various clinical settings including haematopoietic stem cell and solid organ transplant and infectious disease progression. The determination of KIR genes has been used as a factor in the selection of optimal stem cell donors with haplotype variations in recipient and donor giving differential clinical outcomes. Methods to determine KIR genes have primarily involved ascertaining the presence or absence of genes in an individual. With the more recent introduction of massively parallel clonal next-generation sequencing and single molecule very long read length third-generation sequencing, high-resolution determination of KIR alleles has become feasible. Determining the extent and functional impact of allele variation has the potential to lead to further optimisation of clinical outcomes as well as a deeper understanding of the functional properties of the receptors and their interactions with ligands. This review summarizes recently published high-resolution KIR genotyping methods and considers the various advantages and disadvantages of the approaches taken. In addition the application of allele level genotyping in the setting of transplantation and infectious disease control is discussed.

KIR structure and function
The killer immunoglobulin-like receptor (KIR) genes, located on chromosome 19, encode a family of cell surfaceexpressed transmembrane proteins that regulate the killing of virus infected or malignant cells by natural killer (NK) cells and some T cell subsets (Wende 1999). So far, 17 genes have been described which broadly fit into two groups: inhibitory or activating, depending on whether the protein has a long (L) [2DL1, 2DL2, 2DL3, 2DL4, 2DL5A, 2DL5B, 3DL1, 3DL2, 3DL3] or short (S) [2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 3DS1] cytoplasmic tail. In addition, there are two pseudogenes [2DP1,3DP1]. Figure 1 shows a schematic of KIR protein structural organisation. The inhibitory KIR possess an immunoreceptor tyrosine-based inhibitory motif (ITIM) within their cytoplasmic domain (Blery et al. 1997), whereas activating KIR bring about activation via crosslinking with DAP12 a disulphide-bonded homodimer containing an immunoreceptor tyrosine-based activation motif (ITAM) (Lanier et al. 1998). The extracellular Ig domains are designated D 0 , D 1 and D 2 moving from distal to proximal to the cell membrane. The KIR3D receptors possess the D 0 , D 1 and D 2 domains; KIR2DL4 and 2DL5 possess D 0 -D 2 ; all other KIR2D receptors possess D 1 and D 2 . The ligands for inhibitory KIR are known to be autologous HLA-class I molecules and therefore help maintain tolerance to healthy self-tissues.
Historically, KIR genes have mostly been 'typed' to determine their presence or absence within the individual's genome. The KIR genes show a high degree of variation in individual gene content and gene copy number, but are also highly organized. The genes are located in the leucocyte receptor complex (LRC) between LILR and FCAR and sit head to tail with regular spacing. Framework genes KIR3DP1, KIR2DL4, KIR3DL2 and KIR3DL3 occur in nearly every individual and flank regions of variability (Wilson et al. 2000). At the haplotype level, KIR haplotypes are classified into two groups, namely A and B. Figure 2 shows the gene content of the A and B haplotypes. The KIR A haplotype has been defined as containing KIR3DL3, 2DL3, 2DL1, 2DL4, 3DL1, 2DS4 and 3DL2; whereas the B haplotype shows more variation and has more genes for activating receptors (Uhrberg et al. 1997). An early haplotype model theorized that the centromeric portion of KIR haplotypes segregated into five different types, whereas the telomeric portion segregated into two different types producing ten different 'prototypic' haplotypes, with further variation within these (Hsu et al. 2002). This model sees the centromeric portion as bound by the KIR3DL3 and KIR3DP1 framework genes and can possess KIR2DS2, KIR2DL2 or KIR2DL3, KIR2DL5, KIR2DS3 or KIR2DS5, KIR2DP1 and KIR2DL1. Following KIR3DP1 towards the telomeric end, framework KIR2DL4 delineates the telomeric portion which is then bound by the final framework gene KIR3DL2. This portion can then contain KIR3DL1 with either KIR2DS1 or KIR2DS4; or KIR3DS1 with either KIR2DL5 and KIR2DS3 or KIR2DL5 or KIR2DS5 and either KIR2DS1, KIR1D, or KIR2DS4. Thus, individual gene content varies between as little as eight to as many as 14 KIR genes.
More recently, advances in gene sequencing technology has stimulated much interest in allele level KIR gene sequencing, and the genes also show a high degree of allelic polymorphism. As of December 2020, there were a total of 1532 alleles coding for 668 different proteins described in the Immuno-Polymorphism Database (Robinson et al. 2015). All KIR genes show a degree of allele polymorphism with the highest number of alleles having been described in -3DL1 (183), -3DL2 (165) and -3DL3 (228). KIR allele variation has the effect of further diversifying KIR haplotypes possessing the same set of genes (Shilling et al. 2002). A recent large study of European subjects which defined KIR genes to five-digit resolution showed that certain KIR alleles were in strong linkage disequilibrium (LD), for example KIR2DL4*00501 was in complete LD with KIR3DS1*013 and this combination was only ever found in tB01 haplotypes (Amorim et al. 2021). While KIR genes are now known to be also extremely polymorphic, there is a paucity of data associating individual KIR gene polymorphisms with disease or clinical phenotype. Allelic variation is thought to

KIR ligands
HLA class I molecules act as ligands for some inhibitory KIR to provide the signal to inhibit cell activation (Colonna et al. 1993;Storkus et al. 1991;Ljunggren and Kärre 1990;Moretta et al. 1993). The HLA-C molecules as ligands for KIR can be differentiated into two groups based on the amino acid residue present at position 80. An asparagine at this position occurs in the so called C1 HLA-C, whereas a lysine occurs in the C2, and each group comprises half of all HLA-C antigens. KIR2DL2 and KIR2DL3 are highly specific for C1 molecules, whereas KIR2DL1 is specific for C2. KIR3DL1 is specific of the Bw4 epitope carrying HLA-A and -B molecules (Cella et al. 1994), which comprises an isoleucine residue at position 80, whereas an asparagine is not able to signal inhibition. KIR3DL2 binds HLA-A3 and -A11 (Dohring et al. 1996). The non-classical HLA-G has been suggested as a ligand providing inhibitory signalling to KIR2DL4 by fetal-derived trophoblast cells (Rajagopalan and Long 1999). HLA-class I molecules may also act as activating ligands albeit with less avidity. One study showed KIR2DS4 to consistently bind a subset of HLA-A and -C allotypes but with a weaker binding than that shown for KIR2DL1, 2 and 3 (Graef et al. 2009). Similarly, KIR2DS5 has been shown to be a receptor with C2-specificity at variable levels of avidity (Blokhuis et al. 2017). There are a number of reports of KIR being specific for particular peptides bound to class I HLA, for example KIR2DS1 binding HLA-C2 in the presence of specific Epstein-Barr Virus peptides (Stewart et al. 2005) and KIR2DS2 binding to HLA-A*11:01 and a vaccinia viral peptide (Liu et al. 2014). These interactions were shown to be highly dependent on polymorphic positions in the KIR receptor suggesting that allelic variation is likely to have an effect on specificity for ligand and therefore the control of NK cell effector function. The non-classical HLA-F, localized in the endoplasmic reticulum, has been shown to be a high affinity ligand for KIR3DS1 (Garcia-Beltran 2016). Non-HLA molecules have been shown to act as activating ligands, for example, heparan sulfate/heparin glycosaminoglycans as a ligand for KIR2DL4 providing an activating signal to NK cells (Brusilovsky et al. 2013).
NK cell effector function is thought to be controlled, in part, by KIR via the 'missing self' hypothesis (Ljunggren et al. 1990;Moretta et al. 1992). NK cells are inhibited from killing target cells by the presence of their HLA class I ligands. When these ligands are missing through downregulation such as in some viral infected or tumour cells, NK cells then kill the target cell. In the setting of transplantation, lack of the complementary KIR ligand brings about graft NK cells undergo education to acquire functional competence by way of 'licensing' by self MHC molecules. This process involves MHC-specific KIR as well as other inhibitory receptors and leads to two types of self-tolerant NK cells -licensed or unlicensed (Kim et al. 2005). Interaction between KIR and their ligands is thought to be crucial for the licensing program, and NK cells licensed through two different inhibitory receptors may become more potent in their effector functions (Yokoyama and Kim 2006). NK cells are not fully reactive against MHC class I-deficient stimuli unless their inhibitory receptors have recognized self MHC class I molecules prior to this encounter (Anfossi et al. 2006).

KIR content and transplantation
The KIR genes have been implicated in the outcome of haematopoietic stem cell transplant (HSCT). Most studies so far have focussed on gene content and the presence of certain KIR/KIR ligand mismatches and their effect on leukemic relapse. Mismatch at KIR ligands between host and donor was associated with a significantly reduced risk of relapse in haploidentical transplants in patients with acute myeloid leukaemia without leading to an increase in graft versus host disease (Ruggeri et al. 2002). Transplants using a donor with one or two KIR B haplotypes have been associated with significant improvements in overall and relapse free survival (Cooley et al. 2008). The clinical benefit of a B/x donor was not found to depend on the presence of either KIR2DL2 or KIR2DS2, or any other B-haplotype defining KIR gene. The presence of specific activating KIR genes has been associated with better HSCT outcomes. KIR2DS1 was associated with a decreased rate of AML relapse in donors with HLA-C1/C1 or C1/C2 (Venstrom et al. 2012); however, a recent large study failed to reproduce such an effect , highlighting the issue of often contradictory findings by studies, potentially caused by heterogeneity in transplant protocols, patient characteristics and disease states. Increased KIR alloreactivity has been shown to result in lower relapse and increased survival in unrelated cord blood allo HSCT by some groups and conversely with increased graft versus host disease and risk of death by others. These studies have led to the combination of KIR and HLA genotyping in the selection of stem cell donors. Discussion of the selection of unrelated HSCT donors based on HLA and KIR genotyping is outside of the scope of this article but is reviewed by Wright (2020).
Therefore, determining the presence or absence of KIR genes and KIR haplotype content has been a routine in acute myeloid leukaemia and clinical HSCT for many years; however, the role of individual KIR gene polymorphisms in HSCT and other immunopathologies has not been well studied. Recent sequencing advances, particularly with next-generation sequencing (NGS) technology, has therefore stimulated much interest in allelic level KIR sequencing and the possible association of KIR polymorphism with clinical disease and outcome studies.

KIR genotyping
KIR genotyping methods for the last two to three decades have focussed on determining the presence or absence of KIR genes, which goes someway to determining the haplotypes present in an individual (Fig. 2). Various approaches have been applied such as polymerase chain reaction using sequence specific primers (PCR-SSP) (Uhberg et al. 1997) and PCR using sequence-specific oligonucleotide probes (SSOP) (Crum et al. 2000). Various cloning and sequencing methods have been described (Gardiner et al. 2001) to subtype KIR gene alleles; however, these are generally low throughput and time consuming. These techniques typically focus on very limited regions of sequence and as such provide very limited amounts of information about the degree and relevance of variation at the allele level. More recently next-generation (NGS) and third-generation sequencing (TGS) methods have enabled very high throughput cost effective sequencing of much larger regions -typically whole genes, haplotypes and even whole exome sequencing, which has revolutionized gene analysis. The determination of full-length sequences, including non-coding regions has provided deeper insight into gene structure as well as degree of variation, which can now be applied to functional aspects of the proteins encoded. Short-read length NGS incorporates massively parallel sequencing of clonally amplified target DNA strands to produce millions of reads for analysis; long-read TGS is able to sequence single molecules of DNA along stretches in excess of several kilobases making it highly amenable to resolving complex gene systems with long repetitive elements and structural variations (Goodwin et al. 2016). Long-read platforms may be particularly useful for discriminating the presence of fusion KIR genes created by deletions and recombination during chromosomal rearrangements. The creation of these genes may lead to the shuffling of binding and signalling domains thus altering the response of these functional distinct gene variants (Bruijnesteijn et al. 2020). Table 1 summarizes the next-generation sequencing methods discussed below. This review will now focus on these recently developed methods in their application to high-resolution KIR gene sequencing.

Target capture
A target capture sequencing approach has been described where libraries of sheared genomic DNA were enriched for the KIR region using a set of capture oligonucleotide probes prior to NGS using the Illumina platform (Norman et al. 2016). The KIR probes are non-overlapping 80-mers designed from a panel of reference KIR haplotypes representing 13 recognized KIR haplotypes. The probes are biotinylated and purified by binding to streptavidin-coated beads. DNA samples are sheared to 800 bp fragments and individually barcoded using Illumina dual indexes before pooling. Target capture is carried out by hybridising the pooled library to the capture probes and using streptavidin beads to separate from non-target un-hybridized DNA. This workflow was used to sequence libraries from: cell lines of the International Histocompatibility Workshop Group (IHWG); families from West Africa, 15 individuals from the KhoeSan population and exome sequences from 2112 individuals from the 1000 Genomes project. Gene content had been previously determined in the IHWG samples by PCR, and KIR alleles were previously determined in the KhoeSan samples by pyrosequencing and Sanger sequencing. An inhouse bioinformatics pipeline called PING was developed which harvests KIR-specific reads for passing through modules for gene content identification, copy number determination and allele genotyping. Complete concordance was achieved with the known gene content of the IHWG samples. The analysis of 13 KIR genes in the 97 IHWG identified 144 different KIR sequences including 16 novel alleles, which were subsequently confirmed by alternative methods. KIR copy-number and allele data in the KhoeSan samples matched that previously described. From the analysis of the 1000 Genomes data, 100 novel KIR alleles were identified, which were subsequently confirmed from the genomic DNA using Sanger sequencing. One of the main benefits of the method described is the use of capture probes to obtain a sequencing target instead of PCR amplification. This abrogates PCR error and the risk of allele drop out and amplification bias. The authors state that short-read technology was chosen due to its high fidelity; however a disadvantage of short-read sequencing technology is that many of the reads would map to more than one locus due to the high degree of sequence homology in the KIR genes, these reads would then be discarded during analysis. In addition, phasing of polymorphisms could be less reliable leading to ambiguous results. The authors suggest that the method should be able to be applied to longer-read length sequencing chemistry such as Pacific Biosciences or Oxford Nanopore. This KIR genotyping workflow was recently employed to analyse a cohort of 2130 healthy individuals of European descent (Amorim et al. 2021). This study has revealed far more gene content and structure variation than any other work to date, particularly among the framework genes, with, for example, more than 6% of individuals carrying a deletion or duplication of KIR2DL4. The PING pipeline has recently been refined and updated , improving the throughput and enhancing copy number determination. Read misalignment with the highly homologous KIR2DS1 and KIR2DL1 genes was also improved as was the need to interpret unresolved genotypes. Even with these improvements, this study clearly highlights the difficulty in assigning reads by a bioinformatic pipeline. Misaligned reads were most frequently seen in the KIR3DP1 gene, which received reads originating from KIR3DL3, KIR3DL2, KIR2DP1 and KIR2DL4. Pairing of genes where misaligned reads were frequently seen was identified as KIR2DL1 and KIR2DS1 and KIR2DL2 and KIR2DS2. The use of short-read technology almost certainly contributed to the difficulty in aligning reads, for example, in this study, it was observed that 82% of distinct 150-mers were shared between KIR2DL5A and KIR2DL5B. It will be interesting to see this workflow applied to long-read technology.

Long-range PCR
A long-range PCR based assay, followed by Illumina NGS, was described using six primers in a single multi-plex PCR mixture to amplify all full length KIR genes (Maniangou et al. 2017). The amplicons generated varied in size from 4 to 5 kb for KIR3DP1 to 9 to 17 kb for all other genes. Library preparation and dual indexing was performed for sequencing on a MiSeq instrument. A manual bioinformatics pipeline was employed to map sequencing reads to a reference sequence; exon sequences were extracted and compared for polymorphic bases with those contained in the IPD-KIR database. Two different bioinformatic pipelines were then employed for determining KIR allele assignment. This workflow was applied to 30 IHWG DNA samples as a validation. In all 30 samples good coverage was achieved with an average coverage of 316.55X. Allele assignment was obtained for all loci for most samples. The results were compared to those obtained by Norman's capture-based NGS method with the finding that 100% of allele assignments at the three digit level were concordant in 11 KIR genes (n = 110 alleles); semi-discordance, where one allele of a heterozygous typing was different, was found in ten alleles across six loci; and fully discordant results were found in two cases. The authors state that the described method is easily implemented and possibly cheaper than a capture-based method but stressed the requirement for good quality DNA, high fidelity Taq polymerase and reliable library preparation.

Amplicon-based exon sequencing
A different approach using PCR was described where the KIR genes were amplified to produce two amplicons, one covering exons 1 to 5 and the other covering exons 6 to 9 (Closa et al. 2018). A total of eight primers were used in a single multiplex mixture along with eight primers for class I HLA genes. The GENDX NGSgo kit was used to prepare indexed libraries for sequencing on the MiSeq. This workflow was validated with 186 DNA samples made up of 30 IHWG samples with previously determined highresolution KIR genotypes and 156 local DNA samples previously typed for gene content by PCR-SSO. Analysis of the KIR sequences was performed by mapping reads to hg19 which were visualized with commercial bioinformatics software. KIR gene content was determined using four gene specific virtual probes per locus, which were used to find exact matches in FASTQ sequence files. KIR gene content results were found to be 97.84% concordant with the PCR-SSO result. It was estimated that around 250 samples could be analysed in a single MiSeq run using this method.
Although not intended for KIR allele determination, it was suggested by the authors that this approach could be used for allele level typing when a suitable allele calling programme becomes available. A very high throughout short amplicon PCR NGS strategy was described targeting KIR exons 3-5 and 7-9 in four separate reactions (Wagner et al. 2018). Subsequently, the reactions were pooled for each sample and index and sequencing adaptors added. Then a total of 384 samples were combined with pools of HLA, blood group and CCR5 genes for sequencing on the Illumina HiSeq2500. The inhouse bioinformatics pipeline consisted of determining gene content and copy number and then allele assignment with results given as a genotype list (GL) string. The method was validated using DNA from 93 IHWG samples and a reference set of 360 samples previously typed for KIR at the allelic level. In the IHWG samples, 413 genes were identified correctly as being absent, 224 were identified as being present but no allele called due to either a novel nucleotide or failure to meet quality criteria. Finally, 851 genes were typed at the allelic level and all but three were concordant with the previously reported result. The workflow has been used to type 1.8 million potential HSCT donors at the allelic level providing a useful insight into KIR allele frequencies. Differences were seen in the number of alleles present for each gene. Limited diversity was observed in the activating KIR genes with a single allele occurring at high frequency for some genes. Greater diversity was seen in the inhibitory KIR with evenly distributed frequencies. In a subset of 185,170 samples 5203 sequences were identified with novel positions compared to the reference database highlighting the degree to which diversity in these genes is only just beginning to be revealed. While this method demonstrates the power of high throughput NGS workflow with an impressive number of samples genotyped at very low cost, due to the incomplete coverage of the genes ambiguous results are encountered. This method has been employed recently to identify a set of 551 KIR allele group haplotypes representing 21 KIR copy number haplotypes helping to broaden the understanding of the KIR genes (Solloch et al. 2020).

Commercial whole gene PCR
A PCR-based method was developed for commercial use by the sequencing technology company GenDX (van de Pasch et al. 2018). Nine of the 17 KIR genes were targeted in several group specific PCR mixtures which were then subjected to limited group pooling and then library preparation for Illumina MiSeq sequencing. Resulting sequencing data was analysed using the commercially available NGSEngine software. The authors noted the challenges of KIR copy number variation and high degree of homology hampering data analysis; however, the majority of samples were able to be typed unambiguously.

Long-range sequencing using PacBIO platform
A study from the Centre for International Blood and Marrow Transplant Research (CIBMTR) looking at whole KIR haplotypes described a method using long-read technology on the PacBio platform (Roe et al. 2017). Pools of fosmid libraries targeted for KIR sequence content were constructed to span the entire KIR region in eight individuals. Full lengths of KIR locus inserts were isolated from the linearized fosmids and subjected to SMRT sequencing on the PacBio RS II. Using this approach, continuous reads of around 40 kb allowed the unambiguous assembly of two KIR haplotypes for each of the eight diploid samples. Nine distinct haplotype structures were identified in the study samples including four haplotypes not previously described. This method has recently been updated for application to population scale and clinical studies (Roe et al. 2020), replacing the technically demanding fosmid library creation with capture probe enrichment of long fragments of KIR targets which are subsequently sequenced by PacBio. Again, fully sequenced and phased diploid KIR haplotypes with fully annotated alleles were determined.

CRISPR-Cas9 target enrichment
A CRISPR-Cas9 target enrichment method followed by Oxford Nanopore sequencing has recently been described (Bruijnesteijn et al. 2021), where guiding crRNA were designed to direct Cas9 endonuclease to cleave large overlapping DNA segments of the entire KIR region. Cleaved target sites were left with an available phosphate group which was utilized for dA-tailing and then ligation to nanopore sequencing adaptors. Nanopore sequencing relies on a signal change in the electric current passed across a synthetic nanopore as nucleotides pass through the pore. The signal produced is subsequently converted into nucleotide sequence resulting in reads potentially in excess of 100-kb long. This method was used to generate fully phased haplotypes in two subject DNAs, with a third fully phased except for one KIR gene.

Whole KIR gene sequencing on nanopore platform
Our group is developing a full gene PCR amplicon-based method using the Oxford Nanopore Technology MinION platform. Using previously published PCR primers, all KIR genes are amplified and then subjected to a straightforward library preparation involving end repair, barcoding, pooling of around 24 samples, adaptor ligation and loading on to a MinION flow cell. The major challenge that exists for all the described methods is the accurate analysis of genes with a high degree of sequence homology, particularly where throughput gains of multiplexing and sample pooling are highly desirable. Initial analysis of the long-read length output using GENDx NGSEngine in a set of IHWG DNA samples shows promise. This method will be published separately once fully validated.

KIR in HSCT
High-resolution KIR genotyping has been employed in a growing number of studies into the allelic variation present in the KIR gene family. KIR gene allelic variation has been linked with the outcome of HSCT and solid organ transplant illustrating the need for further study of these genes at this level. Allelic variation in KIR genes allows for the characterisation of individuals by differing levels of expression, with high and low expression alleles providing stronger and weaker inhibitory signals. Donors with low inhibition KIR-KIR ligand combinations have been associated with lower relapse and higher survival, whereas combinations with higher expression, higher inhibitory activity were associated with higher relapse (Schaffer and Hsu 2016). Bari et al. have shown that allelic polymorphism in KIR2DL1 affects overall recipient survival and progression-free survival in pediatric allogeneic HSCT patients (Bari et al. 2013). Patients who received grafts with the functionally stronger KIR2DL1 allele with arginine at amino acid position 245 (KIR2DL1-R245) had better survival and lower cumulative incidence of disease progression than those patients who received a donor graft that contained only the functionally weaker KIR2DL1 allele with cysteine at the same position (KIR2DL1-C245). KIR3DL1 alleles have been segregated based on patterns of strong (*001, *002), weak (*005, *007) or lack (*004) inhibition by target cells with HLA-B subtypes and in AML patients who received HLA-compatible allografts, donor-recipient KIR3DL1/HLA-B subtype combinations that demonstrate weak or no inhibition were associated with significantly lower relapse and higher survival compared with strong inhibition combinations (Boudreau et al. 2017). A pilot study found that weak or non-inhibiting KIR3DL1 subtype donors could be identified for 93% of 211 patients who had more than one donor available, and that patients with weak or non-inhibiting KIR3DL1/HLA-B partnerships experienced higher 2-year disease free survival (Schaffer et al. 2016). Subsequently KIR allele subtyping has been shown to be feasible in prospective HSCT donor selection where KIR3DL1 alleles of potential donors were differentiated into high and low expression groups (Schaffer et al. 2021). Donors were thus assessed for KIR advantage with strength of inhibition of donor KIR3DL1 by recipient HLA-B as the priority, followed by presence of HLA-C1/ KIR2DS1 and centromeric KIR haplotype content. In AML patients transplanted with donors with KIR2DL1*003 and KIR2DL3*001, seen in Cen AA individuals, a more consistent and effective NK cell response was seen which led to a significant reduction in the rate of relapse after T-replete haplo-identical HSCT (Dubreuil et al. 2020). A recent study has shown that characterising individuals for the presence of a particular B haplotype defined by allele level genotyping showed a trend to significance for protecting from relapse (Guethlein et al. 2021). In this study, allele level typing was used to differentiate Cen B/B haplotypes into Cen B01 and Cen B02, the latter of which was implicated with relapse protection.

KIR in solid organ transplantation
The role of NK cells has been studied in the solid organ transplant setting. NK cells are known to infiltrate kidney allografts and occur at a higher frequency in the peripheral blood of patients with acute graft rejection. A role for inhibitory KIR-KIR ligand has been shown in the increased risk of chronic rejection and reduction long-term graft survival (Littera et al. 2017). KIR2DS4 and KIR2DS5 have been implicated in the outcome of renal transplantation in patients with glomerular nephritis (Nowak et al. 2012). KIR2DS4 was more frequent in patients with acute graft rejection, whereas KIR2DS5 was associated with decreased rejection. Interindividual differences in KIR and HLA class I ligand genotypes associated with differences in NK cell reactivity impact donor-specific antibody-mediated NK cell antibody dependant cell-mediated cytotoxicity in organ allografts (Rajalingam 2016). In addition, differences have been shown in the number of KIR2D expressing CD8 + T cells between liver transplant recipients with and without acute rejection, suggesting a role for KIR receptors in liver allograft outcomes (Lopez-Alvarez et al. 2011).

KIR in HIV infection and control
In the context of infectious disease, KIR molecules have been shown to exhibit an effect in a variety of settings. The protection from HIV disease progression was shown to be associated with the presence of KIR3DL1 and its ligand HLA-B Bw4, particularly where the 80I is present (Martin and Gao et al. 2002). More recently, in individuals possessing HLA-B*57, a variant of the KIR3DL1 molecule has been shown to be associated with elite control of viral load and delay in disease progression (Martin and Naranbhai et al. 2018). The presence of a valine residue at position 47 of the KIR molecule was significantly higher in elite controllers of the disease. Similarly, KIR2DL3 has been shown to be protective against Hepatitis C virus whereas KIR2DL1 has been associated with recurrent human cytomegalovirus infection.

18th IHIWS workshop: KIR genotyping project
The 18th IHIWS Workshop taking place in Amsterdam, the Netherlands, May 2022 includes a project for sequencing full length KIR genotypes. Laboratories interested in KIR are encouraged to participate and submit samples for KIR sequencing (https:// www. ihiw18. org/ compo nent-immun ogene tics/ kir-typing/).
The goal of this project is to characterize the nature and extent of KIR allelic diversity across human populations using next-generation sequencing (NGS). Participating labs will perform high-resolution KIR genotyping in unrelated individuals from diverse populations. Studies in families will also be conducted in order to define phased KIR haplotypes through segregation analysis.

Conclusion
Next-generation and third-generation sequencing methods are able to generate a massive amount of sequencing data in a high throughput and cost-effective manner. The super high resolution of sequencing able to be performed will provide a detailed view of the KIR gene and haplotype structures. This knowledge can then be carried into studies of how allelic variation might affect NK cell function, outcomes in HSC and solid organ transplantation and the progression of infectious disease. Such studies are likely to provide a greater insight into how KIR molecules interact with their ligands and how variation affects affinity and specificity of the KIR molecules for their targets. Funding Open Access funding enabled and organized by CAUL and its Member Institutions.